Download Restriction Enzyme digestion of DNA

Restriction Enzyme digestion of DNA - Exercise 8
Objectives
-Understand how Restriction Enzymes digest DNA.
-Know how to construct a pAMP (plasmid) or gel.
-Given the size of fragments, gel, know how to
construct a restriction map.
-Given a restriction map know how to construct a gel.
NOTE: DNA IS Negatively charge
because of the phosphate groups.
• DNA molecules are macromolecules that hold the
genetic information of living organisms. They are
extremely long, double-stranded polymers of
nucleotides.
• The covalent bond joining adjacent nucleotides in
DNA is called a phoshodiester bond.
• The phoshodiester bonds between nucleotides in
DNA molecules are very stable unless they are
physically stretched or exposed to enzymes name
nucleases.
• Enzymes are capable of breaking (hydrolyzing)
phoshodiester bonds in DNA molecules. Nucleases
can be classified into two major groups:
exonucleases and endonuclases.
• Exonucleases: If the enzyme digest nucleotides
from the ends of the DNA molecules.
• Endonuclases: If the enzyme digest nucleotides in
the interior of a DNA molecule.
• Restriction endonuclease: An enzythem that
digest DNA by recognizing specific short sequences
of bases that are called palindromes.
• A special class of endonucleases from a bacteria has been isolated for
this experiment. These special enzymes, termed restriction
endonucleases (RE), digest DNA by breaking bonds only within a specific
short sequence of bases. These base sequences usually ran in size from 48 base pairs but can be as long as 23 base pairs.
• Restriction endonucleases confer an adaptive advantage on bacteria by
digesting foreign DNA usually from an invading bacteriphage (bacterial
virus). The resulting DNA fragments can then be further degraded and
destroyed by exonucleases. These enzymes are used to cut DNA in a
precise and predictable manner. They are extensively useful in gene
cloning, DNA amplification, and many recombinant DNA technologies.
• Restriction endonuclease (RE). This RE are also attained from bacteria. In
a bacteria where we get these enzymes form there protected because if a
virus invades a bacteria cell these endonuclease will chop up the virus
DNA, its like a defense system, so we can isolate these endonuclease for
experiments, but bacteria produce these endonuclease to protect
themselves from foreign DNA entering their cells.
2 Restriction Endonucleases (RE)
• EcoR1 & HindIII. Both of these recognize different
nucleotide sequences.
• Each strand of DNA is cut at the phoshodiester bond
between the G and A bases (indicated by the arrow
signs). Notice that the sequence GAATTC is the same
on both strands when each strand is read 5’ -> 3’. Such
symmetrical sequences are called palindromes (In a
English language a palindrome reads the same thing in
both directions). This enzyme cuts the double strands
asymmetrically, leaving protruding ends. These
protruding bases are referred to as sticky ends aka
compatible cohesive ends.
• EcoR1: EcoR1 recognizes palindrome on DNA, and cuts the
bond between G & A, and G & A. When you do that it opens
your DNA. For example, if you have plasmid and that
palindrome is present once on the plasmid, you’ll get one cut.
• If somewhere else that palindrome is present and you incubate
it with EcoR1, you’ll get another cut. So every time EcoR1
recognizes this palindrome on your plasmid, it will cut through
the DNA. So when it opens up the DNA, may get a couple of
unpaired bases, and those unpaired bases are called sticky ends,
and if you throw some nucleotides from different species, you
can make recombinant DNA.
• Like EcoRI, HindIII also recognizes a
palindromic sequence, AAGCTT, and
produces sticky ends. Sticky ends can
hydrogen bond together other because of
complementary base pairing.
• Recombinant DNA molecules are compose
of DNA fragments from two or more
sources. Not all RE’s produce sticky ends.
Some enzymes cut DNA to produce blunt
ends, as shown here.
• Once the DNA has been digested, the fragments must be separated and
identified. Fragments are separated by agarose gel electrophoresis. Agar
is a large polysaccharide.
• Gel electrophoresis: You put an agorose gel (agrose is a polysaccharide) and
it has spaces, your DNA can move through these spaces, you put a current
against this, the negative end is up, the positive is at the bottom, and
because your DNA has a negative charge, the DNA moves down towards
the positive end.
• Gel is immersed in an ionic buffer. The buffer has a pH above 8.0 DNA at
this pH is negatively charge because the phosphates in the DNA backbone
have lost hydrogen ions.
The dye molecules serve as the indicator of the
movements of invisible DNA molecule through
the gel as an electric current is run through the gel.
The negatively charged DNA will migrate from the
anode to the cathode (negative to positive) along
with the current.
• Separation of the DNA fragments occurs as they
migrate through the network of agarose molecules.
Smaller fragments slip through the network fast than
large molecules. The rate of migration is a function of
fragment size, as well as the density of agarose. The
tightness (concentration of agarose).
• High concentration favor smaller fragments.
• Low concentration favor large fragments.
• Each of migration function of fragment size and density
of agarose. Depending on what conformation a circular
DNA gets, it will run differently in the gel. So not only
does the size of the DNA molecule affect migration
rate, but the configuration of the DNA also affects the
migration rate. The DNA that you will
electrophoresing can exist in three different
conformations.
• 1. Supercoil circular: 1st fastest. When its circular
it becomes twisted and turn and be comes a little
bit shorter in size. Migrates fastest down the gel.
Contains small volume, more compacted.
• 2. Linear: Migrates next fastest down the gel.
• 3. Nicked (relaxed) circular: One strand is intact,
the other is broken and when it is nicked, it
becomes extended. This one is very relaxed and
faces the most difficulty making its way through the
agarose.
Supercoil < Linear < Nicked (relaxed) circular
• In addition to conformation affecting migration rate,
laboratory production of plasmid DNA can be produce
very large molecules that migrate very slowly. Two
possible molecules that can be produced are dimers
and concatemers. A dimer consists of two plasmids
covalently linked in a series end to end. A Concatemer,
for example, might consist of two plasmids with one
hooked through the other but not covalently linked to
each other. If a purified uncut plasmid is applied to a
gel, bands of super coiled plasmid, nicked circular
plasmid, dimers, and concatemer can be observed.
• Dimer: Means that its link together by 2 links
• Concatemer: Mean a whole bunch of plasmids linked
together but not covalently linked to each other.
• pAMP - the plasmid DNA. What we did in the experiment on DNA
restriction analysis is we took pAMP (circle) and incubated the pAMP this
plasmid with different restriction endonucleases.
• From your electrophoresis gel, you can estimate the size of pAMP. You
can also determine if pAMP is circular or linear. Finally, you can use the
gel to draw a restriction map. A restriction map is a physical map of a
piece of DNA showing recognition sites of specific restriction enzymes
separated by lengths marked in numbers of bases. Separated DNA base
on size
• The pattern of DNA bands is characteristic for a specific DNA sample and
the restriction enzymes used to cleave it. A banding pattern can be
referred to as a DNA fingerprint. because it is unique to that particular
DNA (and the combination of restriction fragments).
• We ran a gel to see if we could determine how many DNA fragments you
got. By electrophoresing a series of fragments of known size (DNA ladder)
along with the DNA samples of interest, the sizes of unknown fragments
can be estimated.
• A restriction site is a place where an enzymes cuts DNA, so
there are restriction sites for EcoR1, and for HindIII.
• When constructing the pAMP no restriction site where you
start and where you finish.
• Lane 4: A control to see what uncut plasmid looks like. How
uncut DNA traveled whether they made 1 or 2 pieces. It’s
your plasmid DNA DNA was on tube 4 which acts like a
measurements and acts like a ladder. No enzyme (Lane 4).
• Lane 5: DNA ladder: DNA digest, containing known base
pair lengths compare with fragments in lanes 1-3. You will
run DNAs of known size (DNA ladder) to help you estimate
the size of your DNA fragments. Lane 5 contains DNAs of
known sizes (DNA ladder).
Prokaryotic (Circular) DNA
• DNA from bacteria (both chromosomal DNA and extra chromosomal
plasmid DNA) and viruses is often a closed circle. If you have a circular
DNA, we know that’s Prokaryotic DNA. In Prokaryotic DNA, the number of
fragments will equal the number of restriction sites.
Eukaryotic (linear) DNA
• If you have one restriction site for an enzyme, you would have 2 fragments,
and if you have 2 restriction sites for an enzyme, you would have 3
fragments. In Eukaryotic DNA, the number of fragments is always going to
have one more or one less than restriction sites.
• In Eukaryotic DNA, there’s no reason to see multiply bands in control lane
because Eukaryotic DNA is linear, it doesn’t exist as supercoil, relax, or
multimere so this is a hint in lane 4. So when you have Eukaryotic DNA,
you will not see multiply bands in the control lane.
• Also, just because they show you multiply bands, not every time your going
to have prokaryotic (circular) DNA you get multiply lanes, its only if the DNA
has been damaged into a supercoil.
What’s going to effect the movement of the DNA
(Factors)?
• Size: small pieces migrate faster, farther than
bigger pieces.
• Conformation (shape): Comparing 3 pieces of
DNA that are the same size. Supercoil < Linear
< Nicked (relaxed) circular
• Charge: Charge (+,-)DNA is negative because
of Phosphate groups (anode) to positive
(cathode).
Digestion of pAMP with EcoRI & HindIII
• We incubated our plasmid under several conditions. Those
conditions were that we incubate pAMP.
• Lane 1: EcoR1 - one band
• Lane 2: HindIII - one band
• Lane 3: EcoR1 & HindIII - two bands
• Lane 4: Water - Our control. We got one main band.
• Lane 5: DNA ladder, a tool to measure the size of DNA fragments.
When constructing the pAMP.
There’s no restriction site
where you start and where you
finish the map. You could call
this point the reference point.
Also, all your base pairs
(fragments) have to equal the
total number base pairs of your
plasmid. For example, 6,000
Bp’s in this example.
Starting & Ending Point
Key for pAMP
KEY
Enzyme A: Light green
Enzyme B: Pink
Enzyme C: Orange
Enzyme A
Enzyme A
Enzyme A
Enzyme A
Enzyme B
Enzyme B
Enzyme B
Enzyme C
Enzyme C
Enzyme A + B
Enzyme A + B
Enzyme A + B
Enzyme A + B
Enzyme A + B
Enzyme A + B
Enzyme A + B
Enzyme A + C
Enzyme A + C
Enzyme A + C
Enzyme A + C
Enzyme A + C
Enzyme A + C
Enzyme B + C
Enzyme B + C
Enzyme B + C
Enzyme B + C
Enzyme B + C
Enzyme A + B + C
Enzyme A + B + C
Enzyme A + B + C
Enzyme A + B + C
Enzyme A + B + C
Enzyme A + B + C
Enzyme A + B + C
Enzyme A + B + C
Are the number of fragments correct?
How do you cut and paste DNA?
• Enzymes that cut DNA at specific short
sequence sites
– Restriction enzymes digest DNA
• Blunt end cut
• Asymmetric end cut
• Enzymes that paste complementary DNA
fragments together
– DNA ligase
Using a restriction enzyme and DNA ligase to make recombinant DNA
Restriction fragment analysis by Southern blotting
Characteristic pattern of
bands for each sample
DNA is transferred to paper and
denature to single strands
Entire genome
Probe complementary to
the DNA sequence of
interest
DNA bound to radioactive probe
exposes film
DNA CLONING AND ITS APPLICATIONS
• Most methods for cloning pieces of DNA in
the laboratory share general features, such as
the use of bacteria and their plasmids.
• Cloned genes are useful for making copies of
a particular gene and producing a gene
product.
RESTRICTION ENZYMES
• Restriction enzymes are essentially molecular
scissors that cut DNA at specific nucleotide
sequences.
• They originate from bacteria and function as
a defense system against viral invasion. They
“restrict” viral DNA.
Restriction Enzymes
• Cut DNA at highly specific
points
• Recognize specific
sequences
– Four to seven bases
– Each is unique
• Consistent results
STICKY ENDS
• Most restriction enzymes cut double
stranded DNA in an asymmetrical fashion.
• These cuts leave single stranded nucleotide
overhangs that are competent to hydrogen
bond.
• These overhangs are called “sticky ends”.
AGAROSE GEL ELECTROPHORESIS
• One indirect method of rapidly analyzing and
comparing genomes is gel electrophoresis.
• This technique uses a gel as a molecular sieve
to separate nucleic acids or proteins by size.
Gel Electrophoresis
• Separation of DNA
fragments
• Based on size
Cathode
Power
source
Mixture
of DNA
molecules
of different sizes
Shorter
molecules
Gel
Glass
plates
Anode
Longer
molecules
Different Endonucleases Yield Different Patterns
Taq1 + AvaII
Taq1 + Pst1
E coli clinical isolates
Questions
• 1. What is a nuclease?
• 2. How does an endonuclease differ from
an exonuclease?
• 3. What is a restriction endonucleases?
Write names of some restriction
endouclease.
Questions
•
1. What is a nuclease?
•
DNA held by covalent bond joining adjacent nucleotides in DNA is called a
phosphodiester bond. The phosphodiester bond between nucleotide in DNA
molecules are very stable unless they are physically stretched or exposed to enzymes
name nucleases.
•
Enzyme capable of breaking (hydrolyzing) phosphodiester bonds in DNA molecules
and classified into exonuclease and endonuclease .
•
2. How does an endonuclease differ from an exonuclease?
•
Endonuclease digest DNA by breaking phosphodiester bonds in the interior of DNA
molecule. Exonuclease enzyme digest nucleotides from the ends of the DNA
molecule.
•
3. What is a restriction endonucleases? Write names of some restriction endouclease.
•
Restriction endonucleases are a special class of Endonuclease from bacteria to cut
DNA. EcoRI & Hind III. These are enzymes digest DNA by recognizing specific short
sequences of bases called palindromic.
Questions
• 4. What are 2 restriction endonuclease (RE) that we
used in our lab? Write DNA sequences these RE
recognize. Do they produce sticky ends or blunt ends
when they cut the DNA molecules?
• 5. How does the number of restriction sites relate to
the number of fragments produced for linear DNA or
circular DNA?
• 6. What is palindromic DNA sequence?
Questions
• 4. What are 2 restriction endonuclease (RE) that we used in our lab? Write DNA
sequences these RE recognize. Do they produce sticky ends or blunt ends when
they cut the DNA molecules?
• EcoRI & Hind III. Both produce sticky ends when cut.
• 5. How does the number of restriction sites relate to the number of fragments
produced for linear DNA or circular DNA?
• Eukaryotic DNA, always going to have one more or one less fragment than you
have restriction sites.
• Prokaryotic DNA, the number of fragments will equal the number of restriction
sites.
• 6. What is palindromic DNA sequence?
• Reading from the same thing in both direction to read the sequences bases that
restriction endouclease recognizes. For example, M’adam I’m adam.
Questions
• 7. What is electrophoresis? What does agarose gel
electrophoresis allow us to do?
• 8. What is the chemical nature of agarose?
• 9. What factors effect the migration rate of DNA through
an agarose gel?
• 10. For DNA molecules of equal sizes, how do the different
shapes (conformation) of DNA differ in terms of distance
traveled through an agarose gel?
Questions
• 7. What is electrophoresis? What does agarose gel electrophoresis allow us
to do?
• It’s a gel that allows move fragment of DNA across by attracting DNA,
which is negative (anode) to opposite side (cathode) positive side base on
size, and conformation of DNA. It will migrate with current.
• 8. What is the chemical nature of agarose? Polysacchirde & sea weed.
• 9. What factors effect the migration rate of DNA through an agarose gel?
Size, shape (conformation), and charge.
• 10. For DNA molecules of equal sizes, how do the different shapes
(conformation) of DNA differ in terms of distance traveled through an
agarose gel? Supercoil travels the fastest, follow by linear, & nicked
Questions
• 11. In your pAMP electrophoresis
experiment, why did you run a DNA ladder
(lane 5) and undigested pAMP DNA (lane
4)?
• 12. Write some practical applications for
use of restriction end nuclease?
Questions
• 11. In your pAMP electrophoresis experiment, why did you run a
DNA ladder (lane 5) and undigested pAMP DNA (lane 4)?
• Lane 4 is control of DNA to see what uncut plasmid looks like.
• Lane 5 is DNA ladder: Containing known base pair lengths and use
to compare with fragments in lanes 1-3.
• 12. Write some practical applications for use of restriction end
nuclease?
• LOOK AT SLIDES 85-107 on this presentation.
Applications of DNA Technology
• Diagnosis of disease
– Viral genome detection (HIV)
– Genetic disorders (screen for defective genes –
hemophilia, CF, breast cancer)
• Production of pharmaceutical products
– Insulin for diabetes
• Gene Therapy
– Replace or supplement of a defective gene
DNA technology has revolutionized biotechnology, the manipulation of organisms or
their genetic components to make useful products.
An example of DNA technology is the microarray, a measurement of gene expression
of thousands of different genes.
Manipulation of DNA
• Selective breeding
Cloning of DNA
-Restriction endonucleases
-Vector
-Gel electrophoresis
-PCR
– Domesticated animals
– Dogs
– Corn
• Molecular Approaches
Uses of DNA technology
– Power, precision and speed
– Transfer of one gene
– Transfer between species
-GMO
-Human Disease
-DNA Fingerprinting
-Bioremediation
Bioremediation
-Biological methods dealing with pollution, oil spills, pesticide residues.
-Gene responsible for breakup of harmful products (enzyme) cloned into bacteria.
-Bacteria are seeded into a contaminated area.
Other applications…
• Environmental Uses
– Mining minerals
– Detoxifying wastes (oil, sewage, pollution)
• Agricultural Uses
– Transgenic organisms
• Sheep with better wool
• Pig with leaner meat
– Genetic engineering in plants
• Resistant to disease and spoilage
• Delayed ripening
• Forensic Investigation
– Identifying criminal by DNA fingerprinting
– Paternity tests
Therapeutic Cloning

Therapeutic Cloning
 Creates embryonic stem cells
 Produces
material for organ transplants
 Has been challenged on ethical grounds
Reproductive Cloning
 Reproductive Cloning
Creates living child
 Produces offspring identical to parents
 Has been done in animals, not people

Gene Therapy
DNA Fingerprinting
• Identifies individuals
– Disease prevalence
– Forensics
– Paternity
• RFLP analysis
• PCR amplification
Sickle Cell RFLP
Applications: Detecting mutations
Detection of Sickle-Cell
94
RFLP – Restriction Fragment Length Polymorphism
•
•
•
•
DNA cut with Restriction Enzyme
Gel electrophoresis
DNA hybridization
Compare bands
• Applications: Catching the bad
guys
• DNA fingerprinting
•
• -Cut DNA with Restriction
Enzymes
• -Gel electrophoresis
• -Compare bands
Figure 20.17 DNA fingerprints from a murder case
PCR amplify small amounts of DNA from crime scene
Digest DNA and compare pattern of bands – DNA fingerprint
MEDICAL APPLICATIONS
• One benefit of DNA technology is
identification of human genes in which
mutation plays a role in genetic diseases.
• We don’t really understand a genetic disease
until we know the mutation, how the gene
works, and how the protein product
functions both normally and in the disease
state.
HUMAN GENE THERAPY
• Gene therapy is the alteration of an afflicted
individual’s genes.
• Gene therapy holds great potential for treating
disorders traceable to a single defective gene.
• Vectors are used for delivery of genes into cells.
• Gene therapy raises ethical questions, such as
whether human germ-line cells should be treated
to correct the defect in future generations.
PHARMACEUTICAL PRODUCTS
• Some pharmaceutical applications of DNA
biotechnology:
– Large-scale production of human
hormones and other proteins with
therapeutic uses
– Production of safer vaccines
SOME EXAMPLES OF BIOTECHNOLOGY
PRODUCTS
1. Tissue Plasminogen Activator- dissolves
bloodclots.
2. Human growth hormone.
3. Insulin
4. Blood clotting factor VIII.
5. Recombinant vaccines such as for Hepatitis B.
6. Bovine Growth Hormone.
7. Tissue Growth Factor beta.
8. Platelet Derived Growth Factor.
FORENSIC EVIDENCE
• DNA “fingerprints” obtained by analysis of
tissue or body fluids can provide evidence in
criminal and paternity cases.
• A DNA fingerprint is a specific pattern of bands
of RFLP markers on a gel.
• The probability that two people who are not
identical twins have the same DNA fingerprint
is very small.
• Exact probability depends on the number of
markers and their frequency in the population.
SOME UNUSUAL PLACES FORENSIC SCIENTISTS
LOOK FOR DNA EVIDENCE.
DNA FINGERPRINTS CAN BE USED TO
DETERMINE PATERNITY
ENVIRONMENTAL CLEANUP
• Genetic engineering can be used to modify
the metabolism of microorganisms.
• Some modified microorganisms can be used
to extract minerals from the environment or
degrade potentially toxic waste materials.
AGRICULTURAL APPLICATIONS
• DNA technology is being used to improve
agricultural productivity and food quality.
ANIMAL HUSBANDRY AND “PHARM”
ANIMALS
• Transgenic organisms are made by introducing
genes from one species into the genome of
another organism.
• Transgenic animals may be created to exploit
the attributes of new genes (such as genes for
faster growth or larger muscles).
• Other transgenic organisms are pharmaceutical
“factories,” producers of large amounts of
otherwise rare substances for medical use.
GENETIC ENGINEERING IN PLANTS
• Agricultural scientists have endowed a
number of crop plants with genes for
desirable traits.
• Herbicide resistance.
• Resistance to pests and disease.
• Improved nutrition.
GOLDEN RICE
• Genetically modified to
accumulate beta
carotene (vitamin A).
• Over a million children
a year go blind from
vitamin A deficiency.